Hydroxysteroid dehydrogenases (HSD's) are a family of enzymes which play a pivotal role in the regulation of steroid hormone action. These enzymes catalyze the interconversion of secondary alcohols to ketones in a positional and stereospecific manner on the steroid nucleus and side chain. They require nicotinamide dinucleotide (phosphate), NAD(P).sup.+, as cofactor. For example, 3.alpha.-hydroxysteroid dehydrogenase, (EC 1.1.1.50; 3.alpha.-HSD) catalyzes the reduction of 5.alpha.-dihydrotestosterone (a potent androgenic steroid hormone) to 5.alpha.-androstan-3.alpha.,17.beta.-diol (a weak androgen). In this reaction, a 3-ketosteroid is converted to a 3.alpha.-hydroxysteroid and, as a result, the potency of the steroid hormone is decreased by five orders of magnitude. Other HSD's carry out reactions of similar importance in the regulation of estrogen, progestin and glucocorticoid action. As a family, HSD's represent target enzymes for drug development.
Research groups headed by Dr. Cecil H. Robinson (Johns Hopkins University) and by Dr. Douglas F. Covey (Washington University School of Medicine) have concentrated on the development of steroidal suicide substrates for HSD's. [BBRC, 1981, 101;2, 495-501; Steroids, 1982, 40;1, 109-119; Steroids, 1979, 34;2, 199-206; Biochemistry, 1986, 25;23, 7295-7300]. Suicide substrates are one class of mechanism-based inhibitors which mimic the normal substrate and are transformed by the enzyme's catalytic mechanism to highly reactive alkylating agents which then inactivate the enzyme by forming a covalent bond at the active site. In this manner, the enzyme catalyzes its own destruction. These compounds have the potential to be highly selective since they are innocuous by themselves until they are transformed by the target enzyme.
Based on the known functions of 3.alpha.-HSD, suicide substrates for this enzyme may have a therapeutic use in potentiating the action of androgens and could be used as an adjuvant in androgen replacement therapy. Such therapy is routinely used in the treatment of hypogonadism of pituitary and testicular origin. Androgens are also essential for the maintenance of male fertility. They can be used as anabolic steroids and can act as anti-estrogens.
3.alpha.-HSD and several other hydroxysteroid dehydrogenases have been reported to catalyze the oxido-reduction of non-steroidal substrates. Examples include 3.beta.-HSD from Pseudomonas testosteroni which reduces cyclohexanone as well as bicyclic and tricyclic ketones [Prog. in Hormone Res., 1967, 23, 349-373], 3.alpha.-HSD and 17.beta.-HSD from rat and mouse liver, respectively, which oxidize 1,2-trans-dihydroxy-3,5-cyclo-hexadiene [Biochem. J., 1984, 222;3, 601-611; J. Biol. Chem., 1983, 258;12, 7252-7255], and rat liver 3.alpha.-HSD which reduces aromatic ketones and quinones [Biochem. J., 1984, 222;3, 601-611]. These observations suggest that suicide substrates based on non-steroids could provide an alternative rational approach to inhibitor design.
In the present invention, monocyclic-aromatic allylic and acetylenic alcohols are shown to be highly selective inhibitors of 3.alpha.-HSD. The non-steroidal nature of these compounds, coupled with the fact they are less expensive and more readily synthesized, indicate that they offer distinct advantages over their steroidal counterparts. This approach may be generally applicable for the development of suicide substrates for other hydroxysteroid dehydrogenases.
Certain suicide substrates of this invention based on monocyclic-aromatic allylic and acetylenic alcohols are transformed by 3.alpha.-HSD to monocyclic-aromatic vinyl and acetylenic ketones. These ketones have the unusual property of alkylating the pyridine nucleotide binding site of 3.alpha.-HSD. Therefore monocyclic-aromatic vinyl and acetylenic ketones may represent a general class of affinity ligands for pyridine nucleotide binding sites.
A number of additional properties have been assigned to the purified 3.alpha.-HSD of rat liver cytosol which suggest that suicide substrates for this enzyme may have broader applications. First, the enzyme can function as a hydroxyprostaglandin dehydrogenase and will catalyze the conversion of PGF.sub.2 .alpha. (a non-inflammatory prostaglandin) to PGE.sub.2 (a pro-inflammatory prostaglandin) [BBRC, 1987, 148;2, 646-652]. Second, deduction of the amino acid sequence of the enzyme from its cDNA indicates that 3.alpha.-HSD has close sequence homology with prostaglandin F synthase (PGF-synthase) [J. Biol. Chem, 1991, 266;14, 8820-8825]. PGF-synthase catalyses the conversion of PGH.sub.2 (an unstable endoperoxy-hydroxyprostaglandin) to the primary prostaglandins which mediate symptoms of inflammation. Third, 3.alpha.-HSD is potently inhibited in a competitive fashion by all the major classes of non-steroidal anti-inflammatory drugs (NSAID's) in rank-order of their therapeutic potency. This inhibition occurs at concentrations that are beneath the peak plasma concentrations observed in man [PNAS, 1983, 80;14, 4504-4508]. Fourth, the anti-inflammatory drug-sensitive 3.alpha.-HSD is widely distributed in rat tissues. It is found in high levels in tissues which play a role in the metabolism of prostaglandins, in, for example, the lung, heart, seminal vesicle and spleen. [Biochem. Pharm., 1985, 34;6, 831-835]. Collectively, these data suggest that 3.alpha.-HSD may be an alternative target for anti-inflammatory drugs.
Because 3.alpha.-HSD displays these additional properties, the suicide substrates based on monocyclic-aromatic allylic and acetylenic alcohols of this invention should inactivate hydroxyprostaglandin dehydrogenases and prostaglandin F synthase in a mechanism based manner.
While the monocyclic aromatic allylic and acetylenic alcohols display high selectivity for 3.alpha.-HSD, their selectivity can be further enhanced by coupling them to appropriate functionalities to imitate NSAIDs. Once these coupled compounds are oxidized by 3.alpha.-HSD to the corresponding ketones they have the capacity to alkylate the NSAID binding site in preference to the NAD(P).sup.+ binding site of 3.alpha.-HSD.
The monocyclic-aromatic and coupled ketones of this invention can cause time dependent inactivation of prostaglandin-H synthase (EC 1.14.99.1, PGH-synthase, cyclooxygenase). Cyclooxygenase converts arachidonic acid to prostaglandins which mediate many physiological and pathophysiological processes including inflammation. Cyclooxygenase is a bifunctional enzyme, catalyzing both the oxygenation of arachidonic acid to the hydroperoxy endoperoxide prostaglandin G.sub.2 and the reduction of prostaglandin G.sub.2 to the hydroxy endoperoxide prostaglandin H.sub.2. Cyclooxygenase is the target for a variety of non-steroidal anti-inflammatory drugs. For example, aspirin is Known to inhibit the oxygenase activity of cyclooxygenase by acetylating a single internal serine located within the polypeptide chain.
This invention covers monocyclic-aromatic allylic and acetylenic alcohols, appropriately coupled compounds which imitate NSAIDS, and all enzyme-generated products which mediate enzyme inactivation.
Based upon the known functions of 3.alpha.-HSD, hydroxyprostaglandin dehydrogenases, and prostaglandin synthases (PGF synthase and PGH synthase), inhibitors for these enzymes may have a number of broad uses: (1) they may potentiate androgen action and act as adjuvants in androgen therapy; (2) they may represent suicide substrates of hydroxyprostaglandin dehydrogenases and prostaglandin F synthase; (3) they may act as time dependent inactivators of cyclooxygenase; and (4) they may represent a new class of anti-inflammatory drugs.